Botulinum toxin is a neurotoxin protein produced by the bacterium Clostridium botulinum. This toxin blocks the presynaptic release of acetylcholine at the neuromuscular junction, causing flaccid (sagging) paralysis of muscles in mammals. The toxin has proven to be effective for treating strabismus, idiopathic blepharospasm and hemifacial spasm. In addition, it has recently been found to provide relief for a number of motor disturbances of involuntary muscles, including spasmodic torticollis, oromandibular dystonia, and spasmodic dyphonia. Further, botulinum toxin received FDA approval for temporary improvement in the facial appearance of moderate-to-severe frown lines and for the non-surgical treatment of hyperhidrosis (excessive underarm sweating).
The botulinum toxin proteins serotype A and B are now formulated into dosage forms for use in medical applications such as the treatment of torticollis, blepharospasm, hyperhidrosis, etc. as well as in cosmetic applications such as wrinkle reduction. Protein drugs, including botulinum toxin proteins, however, suffer from many problems during the preparation thereof. The problems, most of which are attributed to protein instability, are particularly pronounced for the protein drugs which are formulated with very low concentrations of active proteins, such as botulinum toxins.
Adhesing themselves onto solid surfaces, botulinum toxin proteins, when incased in vessels, are apt to adhere to the inner walls of the vessels, resulting in a loss of the active ingredient. A stabilizing agent is also required for preventing the proteins from being oxidized or degraded into fragments.
Albumin is selected in most cases for use as a stabilizer in the formulation of botulinum toxin. In addition to stabilizing the active protein ingredients in pharmaceutical compositions, albumin enjoys the advantage of showing negligible immunogenicity even when injected into the body. However, serum products such as albumin are not completely free from the possibility of being contaminated with pathogens or microorganisms and thus acting as mediators of disease, particularly viral diseases or Creutzfeldt-Jakob disease.
Often, gelatin is employed in place of albumin. Gelatin, however, is recommended not to be used as a stabilizer for drug formulation because this protein, similarly to albumin, is also obtained from animals and may mediate diseases.
A stabilizer derived from non-animal sources is provided for the pharmaceutical formulation of botulinum toxin by Korean Patent No. 10-0799400 in which recombinant serum albumin (rSA), produced in yeast, is applied for pharmaceutical formulation. However, complete avoidance cannot be provided to the possibility that a neoepitope, a new antigenic structure, may be generated in the course of the production, isolation and recovery of recombinant serum albumin (rSA), eliciting an immune response from the recipient of the drug. Korean Patent No. 10-0665469 discloses a pharmaceutical composition comprising a botulinum toxin, polysaccharides (including hydroxyethyl starch) and an amino acid such as lysine, glycine, histidine or arginine. This pharmaceutical composition is provided in a dosage form prepared by lyophylization and must unfortunately be stored at low-temperature in a refrigerator or freezer. The requirement that the lyophilized botulinum toxin be thawed or diluted just before use may cause an error in the protein. In addition to being inconvenient for use, lyophilized botulinum toxins are difficult to develop into a pre-filled syringe administration type.
A composition for stabilization of protein agents in pharmaceuticals is suggested by U.S. Patent Publication No. 2007-0134199. The composition comprises a non-ionic detergent, preferably polysorbate, and a combination of either glutamine and glutamic acid or asparagine and aspartic acid. A dilution of botulinum toxin in the liquid composition was observed to be stable for 8 months when stored at 4° C. However, because storage at 37° C. decreased the activity of the dilution within one month, the composition has limited use for the stabilization of protein agents, such as botulinum toxin, in pharmaceuticals to be stored at room temperature. This deficit is, in the opinion of the present inventors, attributed to the presence of the polar amino acids and particularly the acidic amino acids such as glutamic acid or aspartic acid.